Expression sites and developmental regulation of genes encoding (1→3,1→4)-β-glucanases in germinated barley

Expression sites of genes encoding (13,14)--glucan 4-glucanohydrolase (EC 3.2.1.73) have been mapped in germinated barley grains (Hordeum vulgare L.) by hybridization histochemistry. A³²P-labelled cDNA (copy DNA) probe was hybridized to cryosections of intact barley grains to localize complementary mRNAs. No mRNA encoding (13,14)--glucanase is detected in ungerminated grain. Expression of (13,14)--glucanase genes is first detected in the scutellum after 1 d and is confined to the epithelial layer. At this stage, no expression is apparent in the aleurone. After 2 d, levels of (13,14)--glucanase mRNA decrease in the scutellar epithelium but increase in the aleurone. In the aleurone layer, induction of (13,14)--glucanase gene expression, as measured by mRNA accumulation, progresses from the proximal to distal end of the grain as a front moving away from, and parallel to, the face of the scutellum.Abbreviations cDNA copy DNA - RNase ribonuclease     

Monensin inhibits the secretion of α-amylase but not polysaccharide slime from seedling tissues ofZea mays

Summary The effect of monensin on polysaccharide slime secretion by root tips of corn (Zea mays) was studied. Various treatment times and ionophore concentrations were tested: none resulted in inhibition of slime secretion. Because monensin changes the pH of the medium, its effect was also monitored in strongly buffered media and at different pH's. Even in such media, monensin did not inhibit slime secretion. We also measured the effect of the drug after a pulse with [³H]fucose or a pulse followed by a chase. The amount of labeled slimed secreted was not altered by the ionophore. However, 10M monensin affected the development of root tips and drastically reduced their growth. We showed that monensin inhibits the secretion of -amylase by the scutellum of the same plantlet. The importance of the nature of the secretory compound in relation to monensin inhibition of its secretion is discussed.Abbrevations Hepes N-2-hydroxyethylpiperazine-N-2-ethane-sul-fonic acid - Mes 2-(N-morpholino)ethane-sulfonic acid     

Chromosomal location of genes conditioning low amylose content of endosperm starches in rice,Oryza sativa L.

Summary Eight dull mutants that lower the amylose content of rice endosperm as well as waxy mutant and a cultivar with common grains were crossed in a diallele manner. The amylose content of F₁ and F₂ seeds was determined on the basis of single grain analysis. It was concluded that the low amylose content of dull mutants is under monogenic recessive control. Alleles for low amylose content are located at five loci designated as du-1, du-2, du-3, du-4 and du-5. These loci are independent of wx locus located on chromosome 6. The five du loci have an additive effect in lowering the amylose content. Two loci, du-1 and du-4, were found to be located on chromosomes 7 and 4, respectively.     

Genetic diversity among sublines originating from a single somatic hybrid cell of Duboisia hopwoodii + Nicotiana tabacum

Summary The genetic instability of an intertribal hybrid cell line, Duboisia hopwoodii + Nicotiana tabacum, obtained by mechanical isolation of a single hybrid cell was studied. Ten subclones of calli derived from this hybrid cell line were cultured for 3 years, and their genetic makeup clarified as to nuclear DNA content, chromosome constitution, and peroxidase isozymes. Nuclear DNA content differed in each subclone. In most subclones, mean DNA content was lower than the mean DNA content in the original hybrid cell line determined 1 year after fusion. This decrease in DNA content is partly attributable to the elimination of tobacco chromosomes that occurred in all subclones. The extent to which tobacco chromosomes were eliminated varied among the subclones — evidence that chromosome elimination occurred slowly. Peroxidase isozyme analysis indicated the loss of a tobacco-specific isozyme, thus confirming results obtained by chromosome analysis. Shoots regenerated from two hybrid subclones after 2 years were also heterogeneous in morphology and nuclear DNA content.     

Dependence of reaction rate of 5,5′-dithiobis-(2-nitrobenzoic acid) to free sulfhydryl groups of bovine serum albumin and ovalbumin on the protein conformations

The effect of protein conformations on the reaction rate of Ellman's reagent, 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) with sulfhydryl (SH) groups of proteins was examined. The stopped-flow method was applied to follow the reaction of DTNB with SH group of two proteins, bovine serum albumin (BSA) and ovalbumin (OVA), at various concentrations of guanidine hydrochloride and urea. The rates for both the proteins were faster in guanidine than in urea. The rate sharply depended on the protein conformations, which were monitored by changes of helix contents on the basis of the circular dichroism measurements. The reaction rate of DTNB with SH groups of BSA was maximal around 2 M guanidine and 5 M urea. On the other hand, the reaction rate of DTNB with OVA was maximal at 3.5 M guanidine, while it gradually increased with an increase in the urea concentration. The amount of reactive SH group participating in the reaction with DTNB was also estimated by the absorbance change at 412 nm. The magnitudes of absorbance change for the reaction with free SH groups of OVA at low concentrations of the denaturants were appreciably smaller than those for BSA with one free SH group. Most of the four SH groups of OVA might react with DTNB above 5 M guanidine, although only a part of them did even at 9 M urea.     

Solubility and reactivity of caseins and beta-lactoglobulin in protic solvents.

The study of the solubility of unstructured proteins (_S1-, -, and -casein) and well-structured globulin (-lactoglobulin) in low water binary solvent systems demonstrated the crucial importance of solvent polarity and neutralization of protein polar functions on the final outcome of solubility experiments. The solubilities up to 38, 56, and 96% in CHCl₃/CH₃OH (1/1, v/v) acidified with HCl and up to 5, 10, and 25% in CHCl₃/CH₃OH (1/1, v/v) in the presence of triethylamine (TEA) were obtained for -, _S1-, and -casein, respectively. The importance of protein charge neutralization was apparent when the solubilization was performed in basified CHCl₃/CH₃OH media, giving the optimal results when the studied proteins were brought before to their isoionic point. The maximum solubility of -casein at its pI in 30–70% methanol in CHCl₃ was reaching 50–60% with triethylamine (TEA) added. -lactoglobulin could be solubilized up to 70% in CHCl₃/CH₃OH (7/3, v/v) acidified with HCl and up to 40% in CHCl₃/CH₃OH (3/7, v/v) in the presence of TEA. The observed yield of reductive alkylation of -lactoglobulin was much higher (98%) when performed in studied solvent system than in aqueous conditions (75%). Apparently, steric hindrance of the well-folded -barrel (in aqueous conditions) structure masks the portion of -NH₂ groups. In the case of unstructured aqueous media -casein, 90% alkylation yields were obained in organic and aqueous conditions.     

Purification of the Lewis blood-group gene associated α-3/4-fucosyltransferase from human milk: an enzyme transferring fucose primarily to Type 1 and lactose-based oligosaccharide chains

A soluble Lewis blood-group gene associated -3/4-L-fucosyltransferase has been purified from human milk by a series of steps involving hydrophobic chromatography on Phenyl Sepharose 4B, ion exchange chromatography on CM-Sephadex C-50, affinity chromatography on GDP-hexanolamine Sepharose 4B and gel filtration on Sephacryl S-200. The first step separated -3-L-fucosyltransferase activity directed towardsN-acetylglucosamine in Type 2 (Gal1-4GlcNAc-R) acceptors from an -3/4-fucosyltransferase fraction acting on both Type 1 (Gal1-3GlcNAc-R) and Type 2 acceptors. Further purification of this latter fraction on CM-Sephadex and GDP-hexanolamine Sepharose gave a single peak of fucosyltransferase activity that catalysed the addition of fucose toN-acetylglucosamine in both Type 1 and Type 2 acceptors and to theO-3 position of glucose in lactose-based oligosaccharides. The enzyme preparation at this stage resembled previously described -3/4-fucosyltransferase preparations purified from human milk. However, gel filtration of this preparation on Sephacryl S-200 or Sephadex G-150 separated further amounts of -3-fucosyltransferase activity acting solely on Type 2 acceptors and left a residual -3/4-fucosyltransferase that retained strong -4 activity with the Type 1 acceptor, lacto-N-biose 1, and -3 activity with 2-fucosyllactose, but had relatively little -3 activity withN-acetyllactosamine and virtually no capacity to transfer fucose to glycoproteins withN-linked oligosaccharide chains having unsubstituted terminal Type 2 structures.     

Expression of c-myc protooncogene in rat lens cells during development,maturation and reversal of galactose cataracts

It is well established that normal patterns of epithelial cell proliferation and metabolism, and of fiber cell differentiation and maturation are essential for the maintenance of transparency in the ocular lens. Several factors, including exposure to high levels of sugars, have been known to result in the compromise of lens transparency. For example, initiation of lens cell damage by galactose induces lens epithelial cells to proliferate. Elevated levels of c-myc mRNA have usually been correlated with rapid cell growth and increased entry of cells into the S phase. Therefore, changes in c-myc mRNA levels may provide an early indication of the stimulation of lens epithelial cells to proliferate and differentiate, which has been postulated to be an early and important event in response to lens cell injury by galactose. By Northern blot hybridization analysis we quantitated c-myc mRNA levels in the lens capsule epithelia of rats (1) exposed to galactose, and (2) undergoing a partial recovery from the galactose-induced cell damage. At the onset of lens cell damage, we find c-myc mRNA to elevate to 6-fold by 24 hr, and by 48 hr decreases to about 3-fold the normal levels. During recovery, c-myc mRNA continues to be expressed at high levels approaching a 10-fold increase by day 12, then decreasing to levels of about 8-fold the control by day 30. The 24 h transitory elevation in c-myc mRNA in lens epithelial cells is in accord with our previous observations on the 24 h increase in MP26, crystallin and aldose reductase mRNAs following a high influx of galactose. Therefore, the elevation in c-myc mRNA as well suggest that galactose appears to cause lens cells to undergo an early transitory period of gene induction following the exposure of lens cells to galactose.     

Effect of adrenergic agonists and antagonists on alanine amino transferase,fructose-1:6-bisphosphatase and glucose production in hepatocytes

Using rat hepatocytes we confirmed our previous results that glucagon and -adrenergic agonists increased the enzyme activity of alanine aminotransferase (AAT) and propranolol abolished their effects. Only the enzyme activity was measured and other parameters like quantity of the enzyme or activation due to modification were not looked for. As in perfusion experiment phenylephrine and phenoxybenzamine (-agonist and -antagonist respectively) also increased the AAT activity in isolated rat hepatocytes and propranolol reversed these effects. The additive effect of glucagon and phenoxybenzamine on AAT was also persistant in hepatocyte system.Fructose- 1:6-bisphosphatase (Fru-P₂ase), another key enzyme in gluconeogenic pathway, was elevated by glucagon and other -adrenergic agonists both in liver perfusion and isolated hepatocyte experiments and was brought back to the normal level by propranolol. In this case also only the enzyme activity was measured and no other parameters were looked for. Unlike AAT this enzyme was not stimulated by phenylephrine or phenoxybenzamine. But AAT and Fru-P₂-ase activities were increased significantly by adenylate cyclase activators like fluoride or forskolin. Thus, it appears that the regulation of fru-P₂-ase by glucagon is purely a -receptor mediated process whereas AAT activation shows a mixed type of regulation where some well known -agonist and antagonists are behaving as -agonists.Results further indicate the presence of phosphodiesterase in hepatocyte membrane which was stimulated by glucagon and brought back to the normal level by propranolol.The different adrenergic compounds stated above, not only modified the activity of the above two enzymes but also stimulated glucose production by hepatocytes from alanine which was in turn abolished by propranolol as well as amino oxyacetate (AOA), a highly specified inhibitor of AAT. This confirm the participation of AAT in gluconeogenesis from alanine in liver. Forskolin and fluoride also increased the glucose production from alanine and showed additive effects with glucagon, phenylephrine and phenoxybenzamine.